Brain oxidative stress in a triple

Free Radical Biology & Medicine 44 (2008) 2051–2057
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Free Radical Biology & Medicine
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Brain oxidative stress in a triple-transgenic mouse model of Alzheimer disease
Rosa Resende a,b, Paula Isabel Moreira b,c, Teresa Proença d, Atul Deshpande e, Jorge Busciglio e,
Cláudia Pereira a,b,⁎, Catarina Resende Oliveira a,b
a
Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal
Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal
Institute of Physiology, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal
d
Department of Neurology, Coimbra University Hospitals, University of Coimbra, 3004-504 Coimbra, Portugal
e
Department of Neurobiology and Behavior, University of California at Irvine, Irvine, CA 92697, USA
b
c
A R T I C L E
I N F O
Article history:
Received 8 December 2007
Revised 29 February 2008
Accepted 18 March 2008
Available online 28 March 2008
Keywords:
Alzheimer disease
3×Tg-AD mouse
Oxidative stress
Lipid peroxidation
Antioxidants
Free radicals
A B S T R A C T
Alzheimer disease (AD) is a neurodegenerative disease which is characterized by the presence of extracellular
senile plaques mainly composed of amyloid-β peptide (Aβ), intracellular neurofibrillary tangles, and selective
synaptic and neuronal loss. AD brains revealed elevated levels of oxidative stress markers which have been
implicated in Aβ-induced toxicity. In the present work we addressed the hypothesis that oxidative stress
occurs early in the development of AD and evaluated the extension of the oxidative stress and the levels of
antioxidants in an in vivo model of AD, the triple-transgenic mouse, which develops plaques, tangles, and
cognitive impairments and thus mimics AD progression in humans. We have shown that in this model, levels
of antioxidants, namely, reduced glutathione and vitamin E, are decreased and the extent of lipid
peroxidation is increased. We have also observed increased activity of the antioxidant enzymes glutathione
peroxidase and superoxide dismutase. These alterations are evident during the Aβ oligomerization period,
before the appearance of Aβ plaques and neurofibrillary tangles, supporting the view that oxidative stress
occurs early in the development of the disease.
© 2008 Elsevier Inc. All rights reserved.
Alzheimer disease (AD) is a neurodegenerative disease characterized by the presence of senile plaques mainly composed of fibrillar
amyloid-β peptide (Aβ) [1] and neurofibrillary tangles (NFTs)
composed of paired helical filaments (PHF) of hyperphosphorylated
tau [2,3]. Plaques and tangles are present mainly in brain regions
involved in learning and memory such as cortex and hippocampus.
These affected regions typically exhibit synaptic and neuronal loss,
with cholinergic and glutamatergic neurons being the most affected
[4].
Whereas the majority of AD patients suffer from the sporadic form
of the disease, there is an inherited familial form caused by rare
mutations in the APP or PS1 gene [5]. However, the neuropathological
features are shared by both sporadic and familiar forms. Aβ can
Abbreviations: AD, Alzheimer disease; Aβ, amyloid-β peptide; t-BHP, tert-butylhydroperoxide; GPx, glutathione peroxidase; GRd, glutathione redutase; GSH, reduced
glutathione; GSSG, oxidized glutathione; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; NBT, nitroblue tetrazolium; NFT, neurofibrillary
tangle; OPT, ortho-phetaldialdehyde; PHF, paired helical filament; SOD, superoxide
dismutase; 3×Tg-AD, triple-transgenic model of Alzheimer disease; TBA, thiobarbituric
acid; TBARS, thiobarbituric acid-reactive substances.
⁎ Corresponding author. Institute of Biochemistry, Faculty of Medicine, University of
Coimbra, 3004-504 Coimbra, Portugal. Fax: +351 239822776.
E-mail address: [email protected] (C. Pereira).
0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2008.03.012
accumulate through overproduction or decreased clearance, leading
to neurotoxicity and cell death. Although the mechanisms through
which Aβ exerts its toxicity remain unclear, it seems that oxidative
stress plays an important role [6,7]. Aβ and oxidative stress are linked
to each other because Aβ produces oxidative stress [8–11], and prooxidants, in turn, increase Aβ production [12,13]. Moreover, several
antioxidants, namely vitamin E and melatonin, were shown to protect
neurons from Aβ-induced toxicity [8–11]. Aβ-mediated oxidative
stress can be due to either an increase in reactive oxygen species (ROS)
production or a decrease in the endogenous antioxidants, namely, in
the activity of antioxidant enzymes such as superoxide dismutase
(SOD) and glutathione peroxidase (GPx) and of nonenzymatic antioxidants such as vitamin E and GSH [14]. Elevated levels of oxidative
stress markers, namely protein carbonyls, thiobarbituric acid-reactive
substances (TBARS), 4-hydroxy-2-trans-nonenal (HNE), 8-hydroxy-2deoxyguanine (8-OHdG), and 8-hydroxyguanine (8-OHG), have been
found in AD brains [15,16] and it has been suggested that oxidative
stress is an early event that contributes to AD pathology before the
appearance of amyloid plaques [17,18].
Recently, Oddo and colleagues developed a new AD mouse model
that harbors PS1 M146 V, APPSwe, and tauP301L mutations [19] and that
progressively develops extracellular senile plaques, intracellular NFTs,
and cognitive impairments [19–21]. Furthermore, this model is crucial
to study the relationship between Aβ and tau pathologies. In fact, it
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R. Resende et al. / Free Radical Biology & Medicine 44 (2008) 2051–2057
has been demonstrated that genetically augmenting tau levels and
hyperphosphorylation in the 3×Tg-AD mouse has no effect on the
onset and progression of Aβ pathology, suggesting that the link between Aβ and tau is predominantly if not exclusively unidirectional
[22]. Moreover, Aβ immunotherapy reduces soluble tau and ameliorates behavioral deficits in old transgenic mice [23,24]. In addition,
Billings and colleagues [25] have demonstrated that spatial training
reduces Aβ and tau pathologies and amielorates the spatial memory
decline.
Given the critical role that oxidative stress plays in the pathogenesis
of AD, the present work was aimed at evaluating the extension of
oxidative stress and the levels of antioxidant defenses in the 3×Tg-AD
mouse that closely mimics AD progression in humans [19,20]. We have
observed increased levels of oxidative stress, in particular, enhanced
lipid peroxidation and decreased levels/activity of both enzymatic and
nonenzymatic antioxidants, in these transgenic mice before the appearance of Aβ plaques and neurofibrillary tangles, supporting the view that
oxidative stress occurs early in the development of the disease.
Experimental procedures
Materials
Reduced (GSH) and oxidized glutathione (GSSG), nitroblue tetrazolium (NBT), GPx and glutathione reductase (GRd), SOD, xanthine
oxidase, hypoxanthine, ortho-phetaldialdehyde (OPT), N-ethylmaleimide (NEM), β-nicotinamide adenine dinucleotide phosphate reduced
form (β-NADPH), and tert-butylhydroperoxide (t-BHP) were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). The Oxiselect HNE–His
Adduct ELISA Kit was purchased from Cell Biolabs (San Diego, CA,
USA). All the other chemicals were obtained from Sigma Chemical Co.
or from Merck kgaA (Damstadt, Germany).
Transgenic mice and brain homogenate preparation
The derivation and characterization of triple-transgenic (3×Tg-AD)
mice have been described previously [19,20]. Briefly, human APP cDNA
harboring the Swedish mutation (KM670/671NL) and human fourrepeat tau harboring the P301L mutation were comicroinjected into
single-cell embryos of homozygous PS1 M146V knock-in mice. The PS1
mice were originally generated on a hybrid 129/C57BL6 background
[26]. Age-and gender-matched nontransgenic and PS1 knock-in mice
were used as controls. Non-Tg, PS1, and 3×Tg-AD mice were obtained
from Dr. Frank LaFerla's laboratory at the Department of Neurobiology
and Behaviour and Institute for Brain Aging and Dementia, University
of California at Irvine. Brain cortices isolated from 3-to 5-month-old
females were frozen and stored at –80 °C before being homogenized in
0.32 M sucrose, 1 mM EDTA,10 mM Tris, pH 7.4. Protein content of brain
homogenates was determined by using the Bio-Rad protein dye assay
reagent.
Measurement of lipid peroxidation
The extent of lipid peroxidation in brain homogenates was determined by measuring TBARS and malondialdehyde (MDA). TBARS levels
were quantified using the TBA assay [27]. Brain homogenates were
diluted two times with 15% trichloroacetic acid, 0.375% TBA, 0.25 M
HCl, and 0.015% 2,6-di-tert-butyl-4-methylphenol and boiled for
15 min. The samples were chilled on ice and centrifuged for 10 min
at 95.5 g in an Eppendorf 5810R centrifuge. The absorbance of the
collected supernatants was then measured at 530 nm using a microplate reader (Spectra Max Plus 384; Molecular Devices). The amount of
TBARS formed was calculated using a molar extinction coefficient of
1.56 × 105M- 1 cm- 1 and expressed as nanomoles TBARS produced per
milligram of protein. The MDA levels were determined by highperformance liquid chromatography (HPLC) [28], using a Gilson HPLC
apparatus with a reverse-phase column (RP18 Spherisorb, S5 OD2).
The samples were eluted at a flow rate of 1 ml/min and detection was
performed at 532 nm. The MDA content was calculated from a standard
curve prepared using the thiobarbituric acid–MDA complex and was
expressed as nanomoles per milligram of protein. As a measure of lipid
peroxidation, the levels of hydroxynonenal–histidine (HNE–His)
protein adducts were also quantified by using the Oxiselect HNE–His
Adduct ELISA Kit (Cell Biolabs, Inc.). The quantity of HNE–His protein
adduct in brain homogenates was determined using a standard curve
containing known amounts of HNE–BSA (0-10 μg/ml).
Measurement of superoxide dismutase activity
The activity of SOD was evaluated using a spectrophotometric assay
described by Flohé and Ötting [29]. After 2 min incubation of 100 μg of
protein in 1.4 ml of phosphate buffer (50 mM K2HPO4 and 100 μM
EDTA, pH 7.8) containing 200 μl 0.025 mM hypoxanthine, 66.7 μl Triton
X-100, and 66.7 μl 0.1 mM NBT, the reaction was initiated by the
addition of 2 μl 0.025 U/ml xanthine oxidase. The reduction of NBT was
measured at 550 nm (V560 UV/Vis spectrophotometer) for 3 min, at
25°C against a blank prepared in the absence of hypoxanthine. The
activity of SOD was calculated using a standard curve containing
known amounts of SOD (0.25-2 U).
Measurement of glutathione peroxidase and glutathione
reductase activities
GPx and GRd activities were determined spectrophotometrically at
340 nm by the analysis of NADPH oxidation [30,31]. The activity of GPx
was measured after a 5-min incubation, in the dark, of 10 μl of each
sample with 100 μl phosphate buffer (0.25 M KH2PO4, 0.25 M K2HPO4,
0.5 mM EDTA, pH 7.0), 100 μl 10 mM GSH, 100 μl 1 unit GRd, and 480 μl
H2O. Then, 100 μl 2.5 mM NADPH and 100 μl 12 mM t-BHP were added
and the absorbance was measured at 340 nm (Jasco V560 UV/Vis
spectrophotometer) for 5 min, with continuous stirring, against blanks
prepared in the absence of NADPH.
For the determination of the activity of the GRd, 200 μl of each
sample was incubated for 30 s with 1 ml phosphate buffer (0.2 M
KH2PO4, 2 mM EDTA, pH 7.0), 100 μl 2 mM NADPH, and 700 μl H2O. The
reaction was initiated by the addition of 20 mM GSSG. After 3 min at
30°C with continuous stirring, the absorbance was measured at
340 nm (Jasco V560 UV/Vis spectrophotometer), against blanks in the
absence of GSSG. Results were normalized for the amount of protein
per sample.
Measurement of glutathione content
Brain levels of reduced and oxidized glutathione were measured
using a fluorimetric assay, according to Hissin and Hilf [32]. Briefly,
1 mg of protein from the brain homogenates was rapidly centrifuged
at 100,000 g (Beckman, TL-100 ultracentrifuge) for 30 min with 1.5 ml
phosphate buffer (100 mM NaH2PO4, 5 mM EDTA, pH 8.0) and 0.5 ml
2.5% H3PO4 (v/v). GSH levels were measured after the addition
of 100 μl of OPT (1 mg/ml in methanol) to 100 μl of the sample
supernatant and 1.8 ml phosphate buffer and incubation at room
temperature for 15 min. For GSSG determination, 250 μl of the supernatant was added to 100 μl of NEM (5 mg/ml in methanol) and
incubated at room temperature for 30 min. Then, 140 μl of this mixture
was incubated for 15 min with 100 μl OPT in 1.76 ml NaOH (100 mM).
Finally, the fluorescence was measured at 420-and 350-nm emission
and excitation wavelengths, respectively. The measurements were
performed in a Perkin–Elmer Luminescence Spectrometer LS 50B. The
GSH and GSSG levels were determined by comparison with linear
standard curves containing known concentrations of GSH or GSSG
(0-1 μg) and results were normalized for the amount of protein per
sample.
R. Resende et al. / Free Radical Biology & Medicine 44 (2008) 2051–2057
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Results
Lipid peroxidation is enhanced in the 3×Tg-AD mice
The extent of lipid peroxidation was evaluated by measuring the
levels of MDA and TBARS, which are products of the oxidative
modification of lipids [16,34]. HNE, another lipid peroxidation marker
[16], structurally modifies proteins, forming stable adducts termed
advanced lipid peroxidation end products. Because His residues are
major targets [35], we have also quantified the levels of HNE–His
protein adducts. Brain homogenates were prepared from the cerebral
cortex of 3-to 5-month-old 3×Tg-AD mice and the levels of MDA,
TBARS, and HNE–His were compared with those determined in agematched PS1 mice and also in non-Tg animals. At this age the 3×Tg-AD
mouse has not yet developed neither amyloid plaques nor NFTs [19,20].
The PS1 mice express mutant PS1 protein at normal physiological
levels in the absence of endogenous wild-type mouse PS1 [26]. As
shown in Fig. 1A an increase in MDA levels occurred in the brains of the
3×Tg-AD mice, but not in the PS1 mice. Similarly, the levels of HNE–His
protein adduct are higher in 3×Tg-AD mice than in non-Tg and PS1
mice (Fig. 1B). A significant increase in the levels of TBARS was
measured in both 3×Tg-AD and PS1 mice (Fig. 1C) compared with that
determined in non-Tg littermates.
Fig. 1. Lipid peroxidation occurs in the brain of 3×Tg-AD mice. Brain homogenates were
prepared from cerebral cortex of 3-to 5-month-old 3×Tg-AD mice and also age-matched
PS1 and non-Tg mice and the extent of lipid peroxidation was evaluated by determining
the production of (A) MDA, (B) HNE–His protein adduct, and (C) TBARS. Data, expressed
as nanomoles per milligram of protein or microgram per milliliter, are means ± SEM of
the values from at least 10 animals. ⁎p b 0.5, ⁎⁎p b 0.01, statistically significant compared
with the control mice. #p b 0.5, statistically significant compared with PS1 mice.
Extraction and quantification of vitamin E
Extraction and separation of vitamin E (α-tocopherol) from brain
homogenates were performed by following a previously described
protocol by Vatassery and Younoszai [33]. Briefly, 1.5 ml sodium dodecyl
sulfate (10 mM) was added to 0.5 mg brain homogenate, followed by the
addition of 2 ml ethanol. Then, 2 ml hexane and 50 μl of 3 M KCl were
added, and the mixture was vortexed for about 3 min. The extract was
centrifuged at 2000 rpm (Sorvall RT6000 refrigerated centrifuge) and
1 ml of the upper phase, containing n-hexane (n-hexane layer), was
recovered and evaporated to dryness under a stream of N2 and kept at
-80°C. The extract was dissolved in n-hexane, and vitamin E content was
analyzed by reverse-phase HPLC. A Spherisorb S10w column
(4.6× 200 nm) was eluted with n-hexane modified with 0.9% methanol,
at a flow rate of 1.5 ml/min. Detection was performed by a UV detector
at 287 nm. The levels of vitamin E were calculated as nanomoles per
milligram of protein.
Statistical analysis
Data were expressed as the means ± SEM of the indicated number
of experiments. Statistical significance was determined by using oneway ANOVA followed by Tukey post hoc tests. The differences were
considered significant for p values b0.05.
Fig. 2. The enzymatic activity of antioxidant enzymes is affected in the 3×Tg-AD mice. In
brain homogenates prepared from 3-to 5-month-old 3×Tg-AD, PS1, and non-Tg mice the
activity of (A) SOD and also of the enzymes of the glutathione redox cycle, (B) GPx
and (C) GRd, was determined spectrophotometrically. Data, expressed as units per
milligram of protein, are the means ± SEM of the values from at least 10 animals. ⁎p b 0.5,
⁎⁎⁎p b 0.001, statistically significant compared with the control mice. #p b 0.5,
statistically significant compared with PS1 mice.
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R. Resende et al. / Free Radical Biology & Medicine 44 (2008) 2051–2057
Discussion
Fig. 3. GSH and GSSG levels and GSH/GSSG ratio are impaired in the 3×Tg-AD mice.
Brain cortical homogenates were prepared from 3-to 5-month-old 3×Tg-AD, PS1, and
non-Tg mice. (A) The levels of GSH and GSSG were determined using a fluorimetric
assay and (B) GSH/GSSG ratio was calculated. Data, expressed as nanomoles per
milligram of protein, are means ± SEM of the values from at least 10 animals. ⁎p b 0.5,
⁎⁎p b 0.01, ⁎⁎⁎p b 0.001, statistically significant compared with the control mice.
The activity of antioxidant enzymes is increased in the brains
of the 3×Tg-AD mice
SOD, GPx, and GRd are three enzymes involved in cellular
protection against damage induced by oxygen-derived free radicals
[14]. The activity of these antioxidant enzymes was measured in brain
homogenates prepared from the cerebral cortex of 3-to 5-month-old
3×Tg-AD mice, age-matched PS1, and wild-type littermates. Unlike
the PS1 mice, a significant increase in the activities of SOD (Fig. 2A)
and of GPx (Fig. 2B) was observed in the brains of the 3×Tg-AD
animals compared with the controls. The activity of GRd, which is
involved in GSH recycling, was not affected in the PS1 or 3×Tg-AD
(Fig. 2C).
Glutathione content is altered in 3×Tg-AD mice
GSH is one of the most relevant cellular nonenzymatic antioxidants [14]. In the 3×Tg-AD mice, GSH levels were significantly
decreased with a concomitant increase of GSSG (Fig. 3A). As a
consequence, the GSH/GSSG ratio determined in brain homogenates
obtained from these mice was lower than that measured in the non-Tg
littermates (Fig. 3B). On the other hand, in age-matched PS1 mice, the
GSH levels were not significantly affected and GSSG deceased in a
significant manner (Fig. 3A). As a result, GSH/GSSG ratio was not
significantly changed in comparison with the non-Tg animals.
Extracellular senile plaques mainly composed of fibrillar Aβ [1] and
NFTs composed of PHF of hyperphosphorylated tau [3] are the major
neuropathological features of AD. Brain areas involved in learning and
memory processes are reduced in size as a consequence of synaptic loss
and neuronal death that seem to be associated with enhanced oxidative
stress [4]. In fact, AD brains exhibit elevated levels of oxidative
stress markers, namely protein carbonyls, TBARS, HNE, MDA, 8-OHdG,
and 8-OHG [4,15,16,34]. HNE readily reacts with proteins and HNE–
protein adducts have been found in AD brains [37]. More recent studies
of patients with amnestic mild cognitive impairment, the earliest
manifestation of AD, show similar patterns of oxidative damage [38].
These observations suggest that oxidative damage to critical biomolecules occurs early in the pathogenesis of AD and precedes pronounced
neuropathologic alterations. Oxidative stress up-regulates BACE expression and activity [39] and increases Aβ levels [13]. On the other hand,
several in vitro [8–10] and in vivo [11] studies have demonstrated that
oxidative stress is involved in Aβ-induced toxicity, which is prevented by
antioxidants, namely vitamin E and melatonin.
In the present study we evaluated oxidative stress in a tripletransgenic model of AD (3×Tg-AD). Epidemiologic studies have
reported the higher incidence of AD in females [40–42], and Schuessel
and colleagues [43] observed a gender-specific higher vulnerability in
female AD patients toward oxidative stress. Taken together, these
results led us to use female mice. The 3×Tg-AD mouse model develops
senile plaques, NFTs, and cognitive impairments in an age-and regiondependent manner that closely mimics the human disease progression
[19–21]. Intraneuronal Aβ is first detected in cortical brain regions,
whereas the most extensive tau immunoreactivity is apparent in the
CA1 region of the hippocampus, progressively affecting neurons in the
cerebral cortex of older animals. Despite equivalent overexpression of
human APP and tau, Aβ pathology precedes tau pathology by several
months. Extracellular Aβ deposits in cortex are apparent by 6 months
of age but tau alterations are not apparent before 12 months of age
[19]. In this work, we addressed the hypothesis that oxidative stress is
an early event in the progression of AD; therefore, we have used 3-to
5-month-old animals that have not yet developed neither Aβ nor tau
pathologies. Even in the absence of these neuropathological hallmarks,
an increase in the extent of lipid peroxidation, an oxidative stress
marker, was observed. These results support other previous studies
that demonstrate a negative correlation between oxidative damage
and Aβ deposition in AD brain [44,45] and support that oxidative stress
is an early event in the development of AD [17,18]. In the 3×Tg-AD mice,
the oligomerization of Aβ starts between 2 and 6 months of age [24],
suggesting that the oxidative stress observed in the 3-to 5-month-old
mice can be initiated by oligomeric Aβ. Recently, De Felice and
colleagues [46] demonstrated that soluble forms of the Aβ peptide, the
amyloid-derived diffusible ligands, stimulate excessive formation of
Vitamin E levels are decreased in the 3×Tg-AD mice
Vitamin E is the most effective lipid-soluble antioxidant that is able
to block the lipid peroxidation chain reaction [36]. As depicted in
Fig. 4, the vitamin E content in 3×Tg-AD mice brain homogenates was
significantly decreased compared with the control animals. In PS1
transgenic animals, the levels of this nonenzymatic antioxidant were
not significantly different from those determined in the controls.
Fig. 4. The brain levels of the lipophilic antioxidant vitamin E are depleted in the 3×TgAD mice. Vitamin E (α-tocopherol) present in brain homogenates obtained from 3×TgAD, PS1, and non-Tg mice was measured by reverse-phase HPLC as described under
Experimental procedures. ⁎p b 0.5, statistically significant compared with the control
mice. Data, expressed as nanomoles per milligram of protein, are means ± SEM of the
values from at least 10 animals. #p b 0.5, statistically significant compared with PS1 mice.
R. Resende et al. / Free Radical Biology & Medicine 44 (2008) 2051–2057
2055
Fig. 5. Oxidative stress is increased in 3×Tg-AD mice. The decrease in nonenzymatic antioxidants such as vitamin E and GSH leads to lipid peroxidation, increasing the levels of MDA,
HNE–His protein adducts, and TBARS. In the face of increased oxidative stress the activity of the antioxidant enzymes SOD and GPx is increased. The activity of GRd is not affected,
leading to a further depletion of GSH levels.
ROS through a mechanism requiring N-methyl-D-aspartate receptor
activation. The higher neurotoxicity exerted by soluble Aβ1-42 in
comparison with fibrillar Aβ suggests that this event can be mediated
by an increase in oxidative stress leading to cell death [47]. The fibrillar
form of the peptide, being less pro-oxidant and cytotoxic, may
preferentially induce toxicity by modulating BACE-1 expression and
activity, increasing the amyloidogenic APP processing, and then
resulting in a further accumulation of Aβ [48]. Soluble Aβ levels in
Tg2576 mice have been directly correlated with increases in H2O2 [48],
suggesting that soluble Aβ may be responsible for its production.
Oxidative stress can be due to either an increase in ROS production
or a decrease in the activity of the antioxidant enzymes such as SOD
and GPx or nonenzymatic antioxidants, namely, vitamin E or GSH [14].
Manganese-SOD detoxifies superoxide anion (O2U−) to give H2O2,
which is then converted into H2O by either GPx or catalase [14]. We
have observed that GPx activity is increased in 3×Tg-AD mice
compared with the nontransgenic animals, which can be a protective
mechanism to neutralize the formation of H2O2 produced by SOD,
whose activity is also increased. Because the GRd activity is not
statistically altered and GPx requires GSH as a substrate, the GSH
levels are decreased and the GSSG levels are increased in 3×Tg-AD
mice compared with controls. Similar results were obtained in cortical
tissue from Tg2576 mice, in which the activities of SOD and GPx are
increased [49]. The expression of SOD as well as of the GPx is higher in
AD brains than in control non-AD brains [15,50].
Vitamin E and GSH, two nonenzymatic antioxidants, were shown
to be decreased in the triple-transgenic model of AD and the levels of
lipid peroxidation markers, namely MDA, HNE–His protein adduct,
and TBARS, were increased. In another AD mouse model, the Tg2576
mice that carry the APP Swedish mutation, lipid peroxidation also
precedes apparent Aβ deposition and increases in Aβ levels [18].
However, oxidative damage in these Tg mice that harbor APP or PS1
mutations appears later than in the 3×Tg-AD mice. Accordingly, in
brain tissue from PS1 M146 L mice, increased levels of oxidative stress
were observed only in aged animals (19–22 months of age) [51]. In this
model, vitamin E reduces lipid peroxidation, and amyloid deposition
[52] and a chronic antioxidant diet can reduce hippocampaldependent memory deficits without affecting Aβ levels or plaque
deposition [53]. Vitamin E-deficient rats, which undergo continuous
oxidative stress, contain dystrophic neuritis analogous to that
associated with the AD senile plaques [54]. PHF are more often
found in neurites with membrane abnormalities indicative of lipid
peroxidation, suggesting that oxidative stress may play a role in the
development of neuritic abnormalities [55]. Because we have not
observed alterations, neither in enzymatic nor in nonenzymatic
antioxidants, in the PS1 knock-in mouse used in this work as a
control, nor in the MDA or HNE–His levels, we can suggest that the
presence of both APP and tau mutations can accelerate the appearance
of oxidative stress markers. Although this hypothesis requires further
investigation, it is supported by several findings. Increased tau
pathology observed in aged homozygous transgenic mice harboring
the P301L tau mutation was revealed in the altered lipid peroxidation
levels and the up-regulation of antioxidant enzymes. Furthermore,
these mice revealed an increased vulnerability of the mitochondria to
Aβ insult, suggesting a synergistic action of tau and Aβ pathologies on
mitochondrial function [56].
The early increase in oxidative stress can contribute to the development of NFTs in the 3×Tg-AD mice, detected at 12 months [20]. The
involvement of oxidative stress and subsequent lipid peroxidation
products in tau phosphorylation has been suggested [4]. Very recently, it
has been demonstrated that mitochondrial oxidative stress causes
hyperphosphorylation of tau in residues that are hyperphosphorylated
in AD [57]. Lovell and colleagues [58] demonstrated a direct link
between oxidative stress and tau phosphorylation in cortical neurons
in culture. Moreover, modifications of tau by 4-HNE promote and contribute to the generation of the major conformational properties
defining neurofibrillary tangles occurring in AD brains [59].
In summary (Fig. 5), using a triple-transgenic mouse model that
progressively develops amyloid plaques and tangles, we demonstrated that the decrease in nonenzymatic antioxidants such as
vitamin E and GSH leads to lipid peroxidation, increasing the levels of
MDA, HNE–His protein adducts, and TBARS. In addition, we observed
an increase in the activity of the antioxidant enzymes SOD and GPx,
providing evidence that neurons mobilize antioxidants in the face of
increased oxidative stress. However, GRd was not affected and thus
GSH levels are further depleted, contributing to oxidative damage to
nucleic acids, proteins, and lipids. This study demonstrated that in
3×Tg-AD mice the oxidative stress occurs earlier than in other
previously studied transgenic animals that carry only APP (Tg2576)
[18,49] or tau mutations (P301L) [56]. The obtained data support the
amyloid cascade hypothesis [60], suggesting that oxidative stress is an
early event in the development of AD and precedes the accumulation
of Aβ in senile plaques and the formation of NFTs. Altogether, this and
other studies [61] suggest that antioxidant therapy may be beneficial
if given at the early stages of the AD development.
Acknowledgment
Rosa Resende is a Ph.D. fellow from FCT (SFRH/BD/11005/2002).
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